Removal of salt from aqueous solutions for metabolomics: targeted salt precipitation

Abstract

Methods for removing salts from a metabolite solution. The methods comprise forming an insoluble silver phosphate salt. Further methods include methods for removing hydrolyzed fluorous compounds. These methods comprise extraction with a fluorous solvent in the presence of a protonation reagent and/or chromatography on a fluorous affinity resin. Methods also include separating lysed cell debris and denatured proteins/disrupted enzymes from a metabolite mixture in a container with a filter, where live cells are grown prior to the lysis, either adherent to the filter or in suspension above the filter. The cells are then lysed in the container.

What is claimed is: 
     
         1 . A method for preparing a solution comprising metabolites, the method comprising:
 reacting a mixture, optionally in the presence of a fluorous solvent, the mixture comprising metabolites and an ionic liquid comprising a phosphate-containing anion and/or a phosphate-containing additive, with a fluorous compound comprising silver cations, and thereby separating the cation of the ionic liquid from the metabolites and obtaining a solution comprising the metabolites and a silver phosphate precipitate.   
     
     
         2 . The method of  claim 1 , wherein the method further comprises a step of removing the silver phosphate precipitate from the solution. 
     
     
         3 . The method of  claim 1 , wherein the phosphate-containing anion is a compound to which one or more phosphate groups are attached. 
     
     
         4 . The method of  claim 1 , wherein the phosphate-containing additive is a compound to which one or more phosphate groups are attached. 
     
     
         5 . The method of  claim 1 , wherein the phosphate-containing anion contains a monophosphate group, diphosphate group, triphosphate group, or any combination thereof. 
     
     
         6 . The method of  claim 1 , wherein the ionic liquid comprises the phosphate-containing additive, and wherein the phosphate-containing additive comprises a monophosphate group, diphosphate group, triphosphate group, or any combination thereof. 
     
     
         7 . The method of  claim 1 , wherein the anion is a mixture of the phosphate-containing counterion with acetate and/or formate. 
     
     
         8 . The method of  claim 1 , wherein the mixture comprises water, acetonitrile, formic acid, fluorous affinity liquid, the ionic liquid with the phosphate-containing anion, and the fluorous anion and the silver cation. 
     
     
         9 . The method of  claim 1 , wherein the mixture comprises a buffer selected from ammonium acetate, ammonium bicarbonate, formic acid, acetic acid, ammonium formate, 4-methylmorpholine, 1-methylpiperidine, triethylammonium acetate, pyrrolidine or any combination thereof. 
     
     
         10 . The method of  claim 1 , wherein the mixture comprises:
 a fluorous solvent selected from a perfluorocarbon (PFC), hydrofluoroether (HFE), and any combination thereof; and/or   an organic solvent selected from acetonitrile, HFE-7100, or any combination thereof.   
     
     
         11 . The method of  claim 1 , wherein the fluorous compound has the following formula (VII):
   [Z 1 —(CH 2 ) m —SO 2 —N( − )—SO 2 —(CH 2 ) p —Z 2 ].M +   (VII)
   wherein: M +  is silver;
 Z 1  and Z 2  are independently a perfluoroalkyl, an alkyl, a substituted alkyl, a perfluoroaryl, an aryl, or a substituted aryl, wherein Z 1  and Z 2  include together a combined total of 8 or more fluorinated carbon atoms; 
 and m and p are independently 0, 1 or 2. 
   
     
     
         12 . The method of  claim 1 , wherein the method further comprises removing a hydrolyzed fluorous compound from the metabolite solution, the method comprising:
 extracting the metabolite solution comprising the hydrolyzed fluorous compound with a fluorous solvent in the presence of a protonation reagent, and thereby lowering a pH of the solution at or below the pKa value of the hydrolyzed fluorous compound, protonating the fluorous compound and obtaining an aqueous phase comprising metabolites and an organic phase comprising the protonated fluorous compound; and   separating the aqueous phase comprising metabolites from the organic phase.   
     
     
         13 . A method for removing a hydrolyzed fluorous compound from a metabolite solution, the method comprising:
 a) extracting the metabolite solution comprising the hydrolyzed fluorous compound with a fluorous solvent in the presence of a protonation reagent, and thereby lowering a pH of the solution at or below the pKa value of the hydrolyzed fluorous compound, protonating the fluorous compound and obtaining an aqueous phase comprising metabolites and an organic phase comprising the protonated fluorous compound; and   b) separating the aqueous phase comprising metabolites from the organic phase.   
     
     
         14 . The method of  claim 13 , wherein the method further comprises:
 c) loading the metabolite solution comprising the hydrolyzed fluorous compound onto a fluorous affinity resin, and thereby binding the fluorous compound to the resin; and   d) eluting the solution comprising metabolites.   
     
     
         15 . The method of  claim 13 , wherein the water-soluble fluorous compound is a fluorous sulfonate; fluorous sulfonamide, or any combination thereof. 
     
     
         16 . The method of  claim 13 , wherein the fluorous solvent is a perfluorocarbon, hydrofluoroether, or any mixture thereof. 
     
     
         17 . The method of  claim 13 , wherein the protonation reagent is hydrochloric acid, hydrobromic acid, boric acid, phosphoric acid, formic acid, carboxylic acid, acetic acid, or any mixture thereof. 
     
     
         18 . The method of  claim 13 , wherein the fluorous affinity resin comprises silicon dioxide derivatized with fluorous carbon chains, a fluorous styrene-based polymer, a fluorous benzyl-based polymer, a fluorous divinyl-benzene polymer, or any combination thereof. 
     
     
         19 . The method of  claim 13 , wherein the elution solvent comprises methanol, ethanol, isopropanol, acetone, acetonitrile, tetrahydrofuran, or any mixture thereof; and wherein the elution solvent optionally comprises one or more from the following: water, a protonation reagent and a polar organic solvent. 
     
     
         20 . The method of  claim 1 , wherein the method further comprises the following steps for:
 a) growing cells in a double-bottom container comprising an internal chamber with a filter bottom, the internal chamber being suspended in an external chamber and the internal chamber being insertable and removable from the external chamber; wherein the cells are optionally adhered to the filter bottom;   b) filtering the cells adhered to the filter bottom to remove growth media;   c) optionally washing the cells with an isotonic solution;   d) lysing the cells by contacting the cells with an ionic liquid in the internal chamber, thereby obtaining a mixture comprising metabolites and the ionic liquid;   e) filtering the mixture through the filter bottom;   f) collecting the mixture in the external chamber; and   g) reacting the mixture according to  claim 1 .